CYP2C19 and CYP2J2 genotypes predict praziquantel plasma exposure among Ethiopian school-aged children

Metabolism of praziquantel (PZQ), a racemic mixture and the only drug approved to treat S. mansoni infection, is mediated by genetically polymorphic enzymes. Periodic school-based mass drug administration (MDA) with PZQ is the core intervention to control schistosomiasis. However data on the impact of pharmacogenetic variation, nutrition, and infection status on plasma PZQ exposure is scarce. We investigated genetic and non-genetic factors influencing PZQ plasma concentration and its metabolic ratios (trans-4-OH-PZQ/PZQ and cis-4-OH-PZQ/PZQ). Four hundred forty-six school children aged 7–15 years from four primary schools in southern Ethiopia who received albendazole and PZQ preventive chemotherapy through MDA campaign were enrolled. Genotyping for common functional variants of CYP3A4 (*1B), CYP3A5 (*3, *6), CYP2C19 (*2, *3, *17), CYP2C9 (*2, *3), and CYP2J2*7 was performed. Plasma concentrations of PZQ, trans-4-OH-PZQ, and cis-4-OH-PZQ were quantified using UPLCMS/MS. Carriers of CYP2C19 defective variant alleles (*2 and *3) had significantly higher mean PZQ plasma concentration than CYP2C19*1/*1 or *17 carriers (p = 0.005). CYP2C19*1/*1 and CYP2C19*17 carriers had higher trans-4-OH-PZQ/PZQ and cis-4-OH-PZQ/PZQ metabolic ratios compared with CYP2C19*2 or *3 carriers (p < 0.001). CYP2J2*7 carriers had lower mean PZQ plasma concentration (p = 0.05) and higher trans-4-OH-PZQ/PZQ and cis-4-OH-PZQ/PZQ metabolic ratios. Male participants had significantly higher PZQ concentration (p = 0.006) and lower metabolic ratios (p = 0.001) than females. There was no significant effect of stunting, wasting, S. mansoni or soil-transmitted helminth infections, CYP3A4, CYP3A5, or CYP2C9 genotypes on plasma PZQ or its metabolic ratios. In conclusion, sex, CYP2C19 and CYP2J2 genotypes significantly predict PZQ plasma exposure among Ethiopian children. The impact of CYP2C19 and CYP2J2 genotypes on praziquantel treatment outcomes requires further investigation.

Globally, about 250 million people are currently infected with schistosomiasis, and 800 million are at risk of infection in endemic areas, mainly in tropical and sub-tropical regions [1][2][3] .Over 90% of the disease burden is from Sub-Saharan African (SSA) countries 4 .In Ethiopia, approximately 38.3 million people live in schistosomiasisendemic areas 5 .Schistosomiasis was first reported in the country in 1934 6 , and children in high transmission areas are the most affected.Repeated exposure to contaminated water with infectious cercariae leads to chronic schistosomiasis, especially in children 7 .In Ethiopia, schistosomiasis remains among the significant causes of morbidity in children 8 .Chronic schistosomiasis can lead to several health complications, including malnutrition, anemia, impaired childhood development, fatigue, exercise intolerance, and poor cognitive function [9][10][11] .

Data collection and preventive chemotherapy
Socio-demographic characteristics, including sex, age, and clinical data such as body weight, height, nutritional status, and infection status, were collected using a case record form through interviews before MDA.Schoolbased MDA with PZQ and ALB 400 mg was given as preventive chemotherapy through an MDA campaign led by the national NTD control public health program coordinators.The study team had no role in the MDA planning, implementation, and administering of the drugs.The dosing of PZQ was based on the WHO dose pole based on height (corresponding to 40 mg/kg body weight) 29 .All children attending primary schools in the two rural districts received MDA irrespective of their infection status.The nutritional status of the participants was assessed by converting the height for age Z score (HAZ) for stunting and BMI for age Z score (BAZ) for wasting using WHO Anthro Plus software 30 .

Stool and blood samples collection
Two weeks before MDA, stool samples were collected for microscopic examination for screening and diagnosis of S. mansoni and soil transmitted helminths infection using Kato-Katz technique.On the MDA Day, whole blood samples for genotyping were collected in EDTA-containing vacutainer tubes from 446 participants and stored at −80 °C.Two hours post-dose, blood samples were collected from each participant in the heparinized tube and centrifuged at 1000 rpm for 10 min, and plasma samples were stored at −80 °C until analysis.Both whole blood and plasma samples were packed with dry ice and shipped to Karolinska Institutet, Stockholm, Sweden, for laboratory analysis.
The plasma sample preparation procedure for quantification of PZQ, trans-4-OH-PZQ and cis-4-OH-PZQ was adapted from Nyla et al. 25 with slight modification.Briefly, 100 µL of plasma sample was added to 300 µL of internal standard solution consisting of 50 ng/mL of rac-PZQ-d11, trans-4-OH-PZQ-d5 and cis-4-OH-PZQ-d5 in a 50:50 mixture (v:v) of acetonitrile: methanol.The mixture was vortexed for 3 min, followed by centrifugation for 20 min at 3220g at 4 °C.Then, 75 µl of the supernatant was diluted with 75 µL MilliQ water and 5 µL was injected into the UPLC-MS/MS system.Standards and quality control (QC) samples were prepared in the same manner by adding 10 µL standard and QC 10× concentrated solutions to 90 µL blank plasma and precipitating as above.
The UPLC-MS/MS method of quantification of the analytes was done as described previously 20,31 .The calibration curves were constructed within the range of 2.4 to 2500 ng/mL for PZQ and cis-4-OH-PZQ, and from 24 to 25,000 ng/mL for trans-4-OH-PZQ, since the levels of trans-4-OH-PZQ were very high in the samples.About 7-9 calibration points were injected twice before and after the samples.The QC samples were injected every 20 samples.The analytes were quantified using the analyte to internal standard integrated peak area ratio with the Mass Lynx application manager Target Lynx (Waters).Trans-4-OH-PZQ d5 was used as an internal standard also for cis-OH-PZQ since their retention times were similar.Quality control samples at 9.8, 78.1, and 1250 ng/ mL were injected at regular intervals during each analysis.

CYP3A4, CYP3A5, CYP2C19, CYP2C9 and CYP2J2 genotyping
Genomic DNA was extracted from whole-blood samples using QIAamp DNA MidiKit (QIAGEN GmbH, Hilden, Germany) as per the manufacturer's protocol.The purity and quantity of the extracted DNA were measured using NanoDrop 2000 (Thermo Scientific, Saveen Warner, Sweden).Genotyping for the common functional variant alleles in genes coding for CYP enzymes relevant for PZQ disposition was performed using allelic discrimination TaqMan genotyping assays (Applied Biosystems, CA, USA) as previously discussed 20,31 .
The genotyping was performed on Applied Biosystems ® 7500 Real-Time PCR Systems (Applied Biosystems, United States).The final volume of the PCR mixture was 10 μL, consisting of 5μL TaqMan fast advanced master mix (Applied Biosystems, United States), 3.5 μL deionized water, 0.5 μL 20× drug metabolizing genotype assay mix and 1 μL genomic DNA.The thermal cycler condition involves an initial step at 60 °C for 30 s, hold stage at 95 °C for 10 min, followed by PCR stage for 40 cycles of 95 °C for 15 s, 60 °C for 1 min and after reading stage with 60 °C for 30 s.

Statistical data analysis
Baseline socio-demographic, clinical, and laboratory parameters were described using means and standard deviations (SD) or medians and interquartile range (IQR) for continuous variables and as percentages for categorical variables.The Chi-square test was used to compare the genotype and allele frequencies between the observed and expected according to the Hardy-Weinberg equilibrium.
A Univariate analysis followed by a multivariate linear regression analysis was performed to identify predictors of PZQ, trans-4-OHPZQ, and cis-4-OH-PZQ plasma concentrations and metabolic ratios.Variables age, sex, baseline S. mansoni infection status, Soil-Transmitted Helminths (STH) infection, S. mansoni-STH co-infection, anemia status, HAZ, BAZ, and the CYP450 genotypes were tested in the linear regression analysis.Variables with p-value < 0.2 from the univariate analysis were included in the multivariate analysis.In all the analyses, p-values were two-sided, and p < 0.05 was considered statistically significant.Statistical analyses were Vol:.(1234567890

Result Socio-demographic characteristics
A total of 446 school children who received the standard PZQ and ALB preventive chemotherapy through MDA to control schistosomiasis and soil-transmitted helminths participated in this study.The median age of the participants was 11 years (IQR = 8-13).Female participants were 51.6%.The proportions of participants with stunting and wasting were 21.9% and 9.3%, respectively.Only 4.3% of the participants were anemic at baseline assessment, as presented in Table 1.

Genotypes and alleles frequencies
There were no significant differences between the observed and expected genotype frequencies according to the Hardy Weinberg Equilibrium.The highest allele frequency was observed for CYP3A5*3 (48.5%) followed by CYP3A4*1B (40.4%), and the lowest allele frequency was 1.6% for CYP2C9*2 (Table 2).

Effect of CYP genotypes on PZQ concentration and metabolic ratios
For assessing the effect of CYP genotypes on PZQ plasma concentration, its major metabolites, and respective metabolic ratios, we categorized all genotypes as extensive metabolizers (*

Predictors of praziquantel plasma concentration
To identify predictors of PZQ plasma concentration, we analyzed log10 converted concentration data using univariate and multivariate linear regression.Variables with p-value ≤ 0.2 in the univariate analysis, such as sex, wasting, CYP2C19 and CYP2J2 genotypes, were included in the multivariate analysis (Table 4).Sex and CYP2C19 remained significant predictors of PZQ plasma concentration in the multivariate analysis.Male participants had a mean increase in concentration of 0.27 ng/ml (95% CI,0.12 -0.42  5).

Discussion
Schistosomiasis remains a major problem in Sub-Saharan Africa, and millions of children living in endemic countries receive periodic school-based mass praziquantel administration to control and halt transmission.Genetically polymorphic CYP enzymes metabolize PZQ, but the impact of genetic variations on drug exposure, particularly in black Africans, the most genetically diverse population, is not well explored.We investigated the effect of genetic variations on plasma concentrations of PZQ, its major metabolites (trans-4-OH PZQ, Cis-4-OH  There are several notable findings from this study: First, there was a significant association of CYP2C19 and CYP2J2 genotypes with PZQ plasma concentration and trans-4-OH PZQ/PZQ and Cis-4-OH PZQ/PZQ metabolic ratios.Carriers of defective CYP2C19 variant alleles (*2 or *3) had significantly higher PZQ plasma concentration and lower metabolic ratio than CYP2C19 extensive metabolizers.On the other hand, CYP2J2*7 carriers had a borderline lower PZQ plasma concentration and significantly higher cis-4-OH PZQ/PZQ metabolic ratio.Second, sex significantly predicted PZQ plasma concentration and trans-4-OH PZQ/PZQ and Cis-4-OH PZQ/ PZQ metabolic ratios.Male participants had higher PZQ plasma concentration and lower trans-4-OH PZQ/ PZQ and Cis-4-OH PZQ/PZQ metabolic ratios compared to females.No significant effect of having infection by S. mansoni or soil-transmitted helminths, or nutritional status (stunting, wasting) or CYP3A4, CYP3A5, or CYP2C9 genotype on plasma praziquantel concentration or its metabolic ratio was observed.This is the first study to explore the impact of genetic and non-genetic factors, including S. mansoni and soil-transmitted helminth infection status in Ethiopian children.
In humans, praziquantel is metabolized primarily by CYP2C19 to 4-OH PZQ, the major metabolite, and to some extent by CYP1A2, CYP3A4, CYP3A5, and CYP2C9 19,20,25 .Genes coding for these metabolizing enzymes are genetically polymorphic.Thus, investigating the effect of genetics on PZQ exposure is important, especially in the genetically diverse SSA population 20,21 , and variations in treatment outcomes have been reported 13 .Moreover, the African continent contributes more than 90% of the global disease burden 4 , and Preventive chemotherapy through MDA campaigns periodically without prior diagnosis to all at-risk children living in endemic areas.The genotype and allele frequencies of CYP3A4*1B, CYP3A5 (*3,*6), CYP2C9 (*2,*3), CYP2C19 (*2,*3,*17), and CYP2J2*7 observed in our study were similar with those of previous studies conducted in Ethiopian population [32][33][34] .
In addition to exploring the impact of genotype in relevant enzymes involved in the intricate metabolic pathway of praziquantel, we investigated the role of CYP2J2 genotype, which has not been previously examined in this context.It is noteworthy that albendazole, co-administered with praziquantel in school-based MDA programs as per the WHO recommendations in endemic areas, is primarily metabolized by CYP2J2 35,36 .With our study participants receiving both praziquantel and albendazole as part of preventive chemotherapy, we aimed to evaluate any potential impact of CYP2J2 genotype.Drug interactions could potentially affect the relationship Furthermore, the relevance of the CYP2J2 enzyme for praziquantel metabolism has not been previously explored.Our finding of a significant impact of CYP2J2 genotype on praziquantel plasma concentration could be attributed to its influence on albendazole metabolism, potentially altering the absorption, metabolism, and transport of praziquantel.Alternatively, CYP2J2 may directly affect praziquantel metabolism, although this aspect requires further investigation.
Our study found a significant association between CYP2C19 genotype and PZQ plasma concentration; a higher PZQ concentration was observed among children carrying defective alleles CYP2C19 (*2 or *3) compared to those with wild type (CYP2C19 *1/*1)) or CYP2C19 *17 carriers.Likewise, the CYP2C19 genotype was also significantly associated with trans 4-OH-PZQ/PZQ and cis 4-OH-PZQ/PZQ) metabolic ratios.CYP2C19 extensive metabolizers had lower metabolic ratios compared to those carrying defective CYP2C19 alleles variant allele (*2 or *3).Similar findings were reported among S. mansoni-infected Tanzanian children who received praziquantel therapy, where the CYP2C19 genotype was significantly associated with PZQ plasma concentration and the trans 4-OH-PZQ/PZQ metabolic ratio 20 .However, the study in Tanzania did not assess cis-OH-PZQ/ PZQ as we did in this study.This finding strengthens the previous evidence that CYP2C19 is a major metabolic pathway for the formation of major PZQ metabolites such as trans-and cis 4-OH-PZQ 20,25,37 .Furthermore, these findings indicate the relevance of the CYP2C19 genotype in determining praziquantel plasma exposure among children receiving PZQ MDA.However, the relevance of CYP2C19 genotype for the safety and efficacy of praziquantel needs to be investigated.
Our study also found a borderline association between CYP2J2 genotype (p = 0.05) and PZQ plasma concentration, where higher PZQ plasma concentrations were observed among CYP2J2 *1/*1 genotypes compared to CYP2J2 *7 carriers.CYP2J2 genotype was significantly associated with both trans-4-OH-PZQ/PZQ and cis 4-OH-PZQ/PZQ) the metabolic ratios, being higher among CYP2J2 *7 carriers than the wild type (CYP2J2 *1/*1).The CYP2J2 *7 variant allele is reported to be associated with increased enzyme activity 34 , which is in line with our findings.A recent study reported the contribution of CYP2J2 genotype for PZQ metabolism though not statistically significant 31 .The significant association of CYP2J2 genotype with variability in PZQ plasma concentration in our study may indicate the contribution of CYP2J2 enzyme for PZQ metabolism.CYP2J2 metabolizes structurally diverse compounds that are also metabolized by CYP3A4, but with differences in regioselectivity 35,38 .In addition to its role in endogenous metabolism, recent studies highlighted the importance of CYP2J2 for metabolizing various drugs, including anti-malarial and anti-tuberculosis that are widely used in Africa 39,40 .Our study also found significant sex differences in PZQ and its metabolite exposure.Male participants had significantly higher PZQ plasma concentration and lower trans-4-OH PZQ/PZQ and cis-4-OH PZQ/PZQ metabolic ratios than females.This observation aligns with previous research indicating higher CYP enzyme activity among females compared to males 41,42 .Consistent with these findings, our study suggests that females displayed lower PZQ plasma concentrations, implying enhanced enzyme activity.Sex differences in drug metabolism stem from a range of biological factors, including variations in hormone levels, enzyme expression, body weight, body mass index (BMI), fat distribution, and other physiological disparities between males and females 43,44 .These differences can lead to variations in drug absorption, distribution, metabolism, and elimination within the body, thereby resulting in distinct pharmacokinetic profiles between males and females.
Although CYP3A4, CYP3A5, and CYP2C9 enzymes were identified as relevant for the metabolism of PZQ in previous studies 18,19,25,26 , our study found no significant association between CYP3A4, CYP3A5, and CYP2C9 genotypes and PZQ plasma concentration, and its metabolic ratios.This finding further suggests that CYP2C19 is a major metabolic pathway for the formation of major PZQ metabolites in humans.In addition, our study identified the CYP2J2 genotype as another possible pathway for PZQ metabolism.

Conclusion
We conclude that plasma concentrations of PZQ and its metabolic ratio display wide inter-individual variability, partly due to pharmacogenetic variations and sex differences.CYP2C19 and CYP2J2 genotypes significantly predict PZQ plasma concentration and its metabolic ratios -trans 4-OH-PZQ/PZQ and Cis 4-OH-PZQ/PZQ.CYP2J2 genotype is significantly associated with PZQ and its major metabolites exposure; therefore, CYP2J2 could be another important pathway for PZQ metabolism in humans.This study highlights the importance of pharmacogenetic variation for PZQ pharmacokinetics.The impact of genetic variations on PZQ treatment outcomes requires further investigations.

Table 4 .
Univariate and multivariate linear regression analysis for predictors of PZQ plasma concentration.Significant values are in bold.

Table 6 .
Univariate and multivariate linear regression analysis for predictors of cis-4-OH-PZQ/PZQ.For the first time, our result indicates CYP2J2 genotype plays an important role in inter-individual variability in PZQ and its metabolites exposure.